Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"

Dr Mohan Z Mani,
Professor & Head,
Department of Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018

Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."

Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018

Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."

Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018

Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."

Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
On Sep 2018

Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."

Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata

Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
On Aug 2018

Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".

Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
On Aug 2018

Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".

Dr. Mamta Gupta
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018

Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.

Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."

Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
On May 11,2011

Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."

Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
On April 2011

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.

Dr. Anuradha
On Jan 2020

Important Notice

Research Protocol
Year : 2023 | Month : November | Volume : 17 | Issue : 11 | Page : EK01 - EK04 Full Version

Expression of PD-1 and PD-L1 in Breast Carcinoma: Research Protocol for a Cohort Study

Published: November 1, 2023 | DOI:
Shreya Ghosh, Jayant S Makarande, Sunita Vagha, Anil K Agrawal, Sahitya Vodithala

1. Junior Resident, Department of Pathology, JNMC, Sawangi, Wardha, Maharashtra, India. 2. Associate Professor, Department of Pathology, JNMC, Sawangi, Wardha, Maharashtra, India. 3. Professor and Head, Department of Pathology, JNMC, Sawangi, Wardha, Maharashtra, India. 4. Professor, Department of Pathology, JNMC, Sawangi, Wardha, Maharashtra, India. 5. Assistant Professor, Department of Pathology, JNMC, Sawangi, Wardha, Maharashtra, India.

Correspondence Address :
Jayant S Makarande,
Associate Professor, Department of Pathology, JNMC, Sawangi, Wardha-442005, Maharashtra, India.


Introduction: Breast cancer is the most common malignant tumour and the leading cause of cancer-related death in women is breast cancer. The Immunohistochemistry (IHC) method utilised antigens and antibodies to interact to identify cellular or tissue constituents (antigens). This research has been employed as a diagnostic tool for specific cancers. When Programmed Cell Death Ligand 1 (PD-L1) binds to Programmed Cell Death 1 (PD-1), it suppresses the cellular immune response by killing and depleting T-cells. Monoclonal antibodies that block the PD-1/PD-L1 pathway have shown promise as a treatment strategy currently being tested in human cancer trials.

Need for the study: Breast cancer is a global issue, and PD-L1 expression is emerging as a promising biomarker for breast cancer prognosis. It can provide valuable information treatment planning.

Aim: The current study aims to examine the expression of PD-1 and PD-L1 in breast cancer using IHC in all subgroups of breast cancer patients. Both tests can serve as a biomarkers to guide immunotherapeutic interventions, improving prognosis, and correlating with other clinicopathological individual parameters such as age, tumour size, distant metastasis, lymph node involvement, Estrogen Receptor (ER) and Progesterone Receptor (PR) status, Her2neu expression, histological type, and TNM stage.

Materials and Methods: This will be a two-year cohort study conducted in the Department of Pathology, Jawaharlal Nehru Medical College (JNMC), Maharashtra, India. The study will include 70 specimens from all cases with a histopathological diagnosis of breast cancer. The Nottingham variant of the Bloom-Richardson Grading System will be used to determine the histological grade of the tumour, and immunostaining for PD-1 and PD-L1 will be performed to evaluate their protein expression.


Breast cancer, Immunity, Programmed cell death 1, Programmed cell death ligand 1

The most prevalent malignant tumour in women and the leading cause of cancer-related death is breast cancer. It is more commonly seen in developed countries (1). Breast cancer has now surpassed cervical and oral cavity malignancies to become the most prevalent cancer and the main cause of cancer death in India (2).

The likelihood of breast cancer in women is higher from their early thirties to their fifties, and the risk continues to rise until it reaches a peak between the ages of 50 and 64 years. Indian women have a one in 28-lifetime likelihood developing breast cancer. It is more common in urban women (1 in 22) compared to rural women (1 in 60) (3). Female breast cancer accounts for 11.7% of all cancer cases, making it the most prevalent type. It ranks as the fifth most common cause of cancer mortality worldwide, with 685,000 fatalities. One in four women is affected by breast cancer, which also causes one in every six cancer deaths (4). Breast cancer can occur due to certain genetic mutations and Deoxyribonucliec Acid (DNA) damage. It has been associated with oestrogen exposure. Some people inherit DNA and gene defects, including those caused by the BRCA1, BRCA2, and P53 genes, among others. Individuals with a family history of ovarian and breast cancer have an increased risk of developing breast cancer. The immune system detects DNA-damaged cells and cancerous cells and eliminates them. Breast cancer occurs when there is inadequate immune defense and surveillance. Disruption of the numerous growth factors and other mediator signaling systems that interact between stromal and epithelial cells can contribute to the development of breast cancer (5).

The two biggest risk factors are gender (99% of those affected are female) and advancing age. Other significant risk factors include early menarche (before the age of 12), late menopause (beyond the age of 55), a late first pregnancy (after the age of 35), nulliparity, the absence of breastfeeding, exogenous hormone therapy, postmenopausal obesity, and inactivity (6).

The most common breast signs and symptoms reported by women with breast cancer are pain, inflammatory changes, nipple discharge, lumpiness, or a palpable mass (6). Although there are many different forms of breast carcinomas, infiltrating ductal carcinoma is the most prevalent histological type (6).

Breast cancer can be detected through various procedures, including breast examination, mammograms, breast ultrasounds, breast Magnetic Resonance Imaging (MRIs), and biopsies. A biopsy is the only reliable method that allows us to make a conclusive diagnosis of breast cancer. The samples are evaluated to determine the cell types that contribute to breast cancer, the degree (grade) of the illness, and whether the cancer cells have hormone receptors or other receptors that could affect the available treatments that are available. Sentinel lymph node biopsy, fine needle aspiration biopsy, core needle biopsy, surgical biopsy, and imaging-guided biopsy are among the numerous forms of biopsy that can be employed (6).

If the tumour is localised, the mainstay treatment for breast carcinoma is surgery, followed by chemotherapy, and radiotherapy. For ER- and PR-positive tumours, adjuvant hormonal therapy, and immunotherapy are also used (7).

The transmembrane glycoprotein death ligand encoded as PD-L1 is found on the membranes of various tumour cells, epithelial cells, and immune cells, including B cells, macrophages, dendritic cells, and T-cells. PD-L1 binds to PD-1, an immuno-inhibitory receptor belonging to the B7-CD28 gene superfamily, which regulates the immune response to tumour cells. When PD-1 on immune cells binds to PD-L1 on tumour cells, T-cell activation is inhibited, leading to reduced T-cell-mediated anti-cancer immunity. Overexpression of PD-L1 has been linked to increased neoplastic development, treatment resistance, and cancer recurrence. Consequently, several immunotherapeutic approaches have been developed. Notably, PD-L1 inhibitor atezolizumab and PD-1 inhibitor pembrolizumab have received Food and Drug Administration (FDA) approval for use in patients with locally advanced or metastatic PD-L1 positive Triple Negative Breast Carcinoma (TNBC) in combination with chemotherapy, indicating the increasing use of immunotherapy in breast cancer treatment (8).

The present study aims to investigate PD-1 and PD-L1 expression through immunohistochemistry (IHC) in relation to clinicopathological parameters such as age, tumour size, distant metastasis, lymph node involvement, ER/PR/Her2neu status, histological type, and TNM stage. The study objectives include determining the histological grade using the Nottingham histological grading system, examining PD-1 and PD-L1 expression in breast cancer tumour tissue using IHC, analyzing the distribution of positive PD-1 and PD-L1 expression in all subtypes of breast cancer patients, and evaluating the potential utility of PD-1 and PD-L1 expression in guiding immunotherapeutic interventions.

Review of the literature

Breast cancer is a global issue, with invasive breast cancer being the most common type of cancer in women worldwide (9). Women’s cancers account for 23% of all cancers globally. There are several genetic markers, such as CD9, CD63, CD81, CD53, and CD37, that may have significant value as prognostic indicators in breast cancer patients. PD-L1 expression is emerging as a promising biomarker for breast cancer prognosis, and it may be useful for doctors in selecting the best treatment for breast cancer. Various grading techniques can be used to determine the grade of breast cancer, with the Nottingham composite histologic grade (Elston-Ellis version of the Scarff-Bloom-Richardson grading system) being one of them, commonly used for reporting. There is a correlation between histologic grade and outcome within each stage grouping. The Nottingham composite histologic grade evaluates factors such as the amount of tubule formation, the degree of nuclear pleomorphism, and the mitotic count.

PD-L1 binds to PD-1 and leads to T-cell death or exhaustion, suppressing the cellular immune response. Monoclonal antibodies that block the PD-1/PD-L1 pathway are a promising treatment strategy currently being tested in human cancer trials (10). These findings suggest that PD-L1 promotes the activation of the PD-1/PD-L1 pathway, facilitating immune escape by tumour cells. PD-L1 expression has been observed in solid tumours such as hepatocellular carcinoma, renal cell carcinoma, testicular cancer, papillary thyroid cancer, breast cancer, lung cancer, gastric cancer, and colorectal cancer. Li CJ et al., reported that breast cancer is a common malignant tumour in women, with triple-negative breast cancer being a particularly aggressive form of the disease with a poor prognosis and a high risk of metastasis. PD-L1 is involved in the evasion of tumour cells from immune surveillance. Significant progress has been made in understanding the biology of triple-negative breast cancer (11). Sun WY et al., found that the detection of PD-L1 in both cancerous and immune cells varied depending on the antibody clone used. PD-L1 (E1L3N) showed the highest staining, followed by PD-L1 (28-8), and then PD-L1 (SP142). Further investigation of the PD-L1 inhibitor clinical trial group is needed to determine the best cut-off value and the gold standard antibody (12).

In a study conducted by Yuan C et al., immunohistochemical techniques were used to determine the positivity or negativity of PD-1/PD-L1. Positive PD-1/PD-L1 cells in metastatic lymph nodes were found to be associated with unfavourable prognostic factors, including a significant number of metastatic lymph nodes (p=0.002), high TNM stage (p=0.012), high Ki-67 index (p=0.048), and high histological grade (p=0.029). It is important to confirm the presence of PD-L1 in both the primary tumour and metastatic lymph nodes due to heterogeneity (13). Han Y et al., reported that cancer immunotherapy has shown promising results in recent years. PD-1 regulates T-cell activity, suppressing immune responses and promoting self-tolerance by preventing the death of regulatory T-cells while inducing it in antigen-specific T-cells (14).

Schütz F et al., found that immunomodulation appears to be a feasible approach in solid tumours. Breast cancer subtypes with high proliferation index, such as TNBC and HER2-positive, have been characterized as highly immunogenic. Immune checkpoints play a role in immune escape, with PD-L1 expression on tumour cells leading to lower CD8+ T-cell activity. Clinical studies are investigating antibodies against PD-1 or PD-L1, and predictive markers are needed to identify patients who would benefit from effective treatment (15). According to Jiang C et al., in addition to T-cell activation, lymphocyte activation, leukocyte migration, T-cell apoptosis, tolerance induction, and cytolysis, genes correlated with PD-1/PD-L1 are also involved in these biological processes. PD-1 expression was associated with immune infiltration, immune regulators, and higher survival rates in patients with breast cancer (16). Bharadwaj KR et al., found that approximately 52% (68/132) of TNBC cases express PD-L1, suggesting that anti-PD-1/PD-L1 therapy alone or in combination with chemotherapy may be a promising treatment for TNBC in Indian patients (17). Alsaab HO et al., noted that not all PD-L1-expressing tumours respond to PD-1/PD-L1 inhibitors, and conversely, PD-L1-negative tumours can respond to these agents. Further studies on this subject are still ongoing (18).

According to Costa R et al., PD-1 is an immune checkpoint inhibitor expressed on immune effector cells. The first PD-1 antibody tested in humans was Nivolumab. It was found that only a small number of patients responded to the treatment, but those who did showed impressive long-lasting tumour remission. However, there were reports of serious immune-related toxicity. Currently, the anti-PD-1 antibody Nivolumab and the anti-PD-L1 antibody Durvalumab are being investigated in breast cancer (19). In a study by Ma W et al., antibodies targeting PD-1 and PD-L1 are showing promising results, but only a subset of patients (20%) respond to immune checkpoint inhibitory treatment. Predictive markers are needed to identify those patients who have the best chance of benefiting from effective treatment (20).

Material and Methods

A cohort study will be conducted for a duration of two years (June 2022 to June 2024) in collaboration between the Department of Pathology and the Department of Surgery at JNMC Maharashtra, India. The study will enroll patients who have been diagnosed with breast carcinoma (IEC No.- DMIMS(DU)/IEC/2022/1070, Date- 27/06/2022).

Inclusion criteria: The study will include all cases with a histopathological diagnosis of breast carcinoma, all specimens of modified radical mastectomy, and all female patients diagnosed with breast carcinoma.

Exclusion criteria: The study will exclude cases of adenomas, malignant lymphomas, myoepithelial lesions, nipple tumours, fibroepithelial tumours, and mesenchymal tumours. Additionally, examples of tumours that can occur in the body such as lactating adenoma, tubular adenoma, pleomorphic adenoma, apocrine adenoma, and ductal adenoma will be excluded. Male patients with breast carcinoma will also be excluded.

The study will involve 70 specimens of breast carcinoma obtained from the Department of General Pathology at JNMC. Formalin-fixed paraffin-embedded blocks will be obtained for analysis. The materials required for the study include 10% formalin, grossing instruments (grossing tray, knife, scalpel, measuring tape, plain forceps, toothed forceps), an automated tissue processing assembly, haematoxylin and eosin stain, PD-1 and PD-L1 markers (PathnSitu), glass slides with dimensions of 7.5 x 2.5 cm, and a binocular research microscope.

Study Procedure

Prior informed consent will be obtained from the patients who will participate in the study. For new patients, a clinical history and physical examination will be conducted, while the clinical details of previously diagnosed cases will be collected, considering the inclusion and exclusion criteria. Biopsies will be taken from clinically suspected cases and sent for histopathological examination at the Department of Pathology, Jawaharlal Nehru Medical College. Routine laboratory tests will be conducted for every patient. The received specimens will be carefully examined and dissected, and appropriate sections will be collected. Normal tissue preparation will be performed before conducting routine Haematoxylin and Eosin (H&E) staining. The histological grade of the tumour will be determined using the Nottingham variant of the Bloom-Richardson Grading System. Immunostaining for PD-1 and PD-L1 will be performed to evaluate the expression of these proteins (21).

Staining Protocol: Haematoxylin and Eosin staining (22),(23): The paraffin-cut sections of breast cancer tumour tissue will be processed using the standard protocol for Haematoxylin and Eosin staining to evaluate histological features.

Immunostaining (22),(23): Formalin-fixed paraffin-embedded tissue sections, with a thickness of four microns, will be placed on slides coated with L-lysine polymer. The slides will be deparaffinized and rehydrated. Antigen retrieval will be performed using a pressure cooker for heat-induced epitope retrieval, followed by three washes with Phosphate Buffer Saline (PBS). Endogenous peroxidase activity will be blocked by treating the slides with 3% hydrogen peroxide for 10 minutes. The PD-1/PD-L1 primary antibody will be applied and allowed to react at room temperature for 30-40 minutes, followed by three washes with PBS. The secondary antibody (streptavidin-biotin) will be applied and allowed to react at room temperature for 30 minutes, followed by another round of PBS washes. The slides will then be soaked in a 3,3-Diaminobenzidine (DAB) solution for 10-20 minutes. After washing with tap water, the slides will be incubated with haematoxylin for 2 minutes at room temperature. The slides will be dehydrated, cleared, and mounted.

Methodology of interpretation: The Nottingham variation of the Bloom-Richardson system will be used to histologically grade the tumour mass (24).

Evaluation of PD-1 and PDL-1 by IHC (25),(26)

PD-1: Positive PD-1 expression will show cytoplasmic and membranous staining in lymphocytes with a cutoff of at least 1%.
a) In tumour cells: Positive PD-L1 expression will be defined as partial or full membranous staining in more than 1% of tumour cells.
b) In immune cells: Positive PD-L1 expression will be defined as cytoplasmic and membranous staining in at least 1% of immune cells.
• A cutoff of >=1% will be considered positive for both PD-1 and PD-L1.

Primary outcome: PD-1 and PD-L1 expression will be studied in all categories of breast cancer patients, and the distribution of positivity will be reported.

Statistical Analysis

A Chi-square test will be conducted to analyse the relationship between PD-1 and PD-L1 protein expression and clinicopathological parameters, including age in years, size in cm, distant metastasis, lymph node involvement, ER status, PR status, Her2neu status, histological type, and TNM stage.


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DOI and Others

DOI: 10.7860/JCDR/2023/60520.18646

Date of Submission: Sep 29, 2022
Date of Peer Review: Dec 07, 2022
Date of Acceptance: Mar 31, 2023
Date of Publishing: Nov 01, 2023

• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

• Plagiarism X-checker: Sep 30, 2022
• Manual Googling: Jan 17, 2023
• iThenticate Software: Feb 25, 2023 (8%)

ETYMOLOGY: Author Origin


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